This proposal describes a series of investigations designed to elucidate the coordination environments of the multiple metal ions in metallothionein and characterize their recently recognized chemical reactivities. Since this sulfhydryl-rich metalbinding protein is a normal cellular constituent in humans and virtually all other organisms and can be induced by the administration of heavy metals, including cadmium, and by a variety of physiological stresses, metallothionein is believed to play an important role in zinc and copper metabolism, heavy metal detoxification, and organismic stress response. A variety of physicochemical techniques will be utilized to elucidate detailed structure-function relationships. Our principal structural tool will be 113Cd NMR, which is a uniquely powerful probe of metallothionein structure and which was recently used to prove that cadmium is grouped in two discrete metalthiolate clusters in the protein. The generality of the two-cluster arrangement will be tested by investigating the 113Cd NMR parameters of metallothioneins from several diverse sources. In concert with 13C NMR studies, the coordination spheres of both cadmium and zinc will be characterized under a variety of conditions. Detailed structural, kinetic and thermodynamic investigations of the formation of metallothionein from metal ions and the apo-protein, thionein, will provide insights into the possible mechanism of its in vivo assembly. Kinetic and mechanistic studies of metal exchange between thionein-bound zinc and cadmium, and the free ions will be carried out, with parallel 113Cd NMR investigation of the differential reactivities of the clusters to metal exchange. Novel reactions of metallothionein including zinc transfer to apo-carbonic anhydrase, EDTA extraction of the metal ions, and disulfide interchange reaction of the thiolates with DTNB will be reinvestigated to determine the relationship of the metal clusters on the reaction kinetics. Novel cadmium complexes with assymetric dithiol chelating ligands will be synthesized as model compounds for 113NMR studies.